Bushen Huoxue formula protects against renal fibrosis and pyroptosis in chronic kidney disease by inhibiting ROS/NLRP3-mediated inflammasome activation

Abstract Background Renal fibrosis contributes to chronic renal failure and a decline in the quality of life. Bushen Huoxue (BSHX) formula is a Traditional Chinese Medicine used to treat chronic renal failure. However, its mechanisms of action remain unclear. Methods and results In this study, a rat model of renal fibrosis was constructed by 5/6 nephrectomy in vivo, and histopathological changes were analyzed using hematoxylin-eosin and Masson’s trichrome staining. Angiotensin II (Ang II) was used to establish an in vitro renal fibrosis cell model in vitro. Pyroptosis was measured using flow cytometry. Related markers of fibrosis and NOD-like receptor protein 3 (NLRP3) inflammasome activation were measured using western blotting and enzyme-linked immunosorbent assay. Treatment with BSHX (0.25, 0.5, and 1 g/kg) significantly inhibited renal fibrosis and damage in 5/6 nephrectomized rats and simultaneously reduced oxidative stress and NLRP3 inflammasome activation. Similarly, BSHX treatment reduced the levels of hydroxyproline, transforming growth factor-β, matrix metalloproteinase 2, and matrix metalloproteinase 9 and inactivated the Smad2/3 signaling pathway in Ang II-treated HK-2 cells. Our data also showed that treatment with BSHX reduced NLRP3 inflammasome activation and pyroptosis in Ang II-treated HK-2 cells. Moreover, fibrosis and pyroptosis in HK-2 cells induced by NLRP3 overexpression were reduced by treatment with BSHX. Conclusions BSHX significantly reduced renal fibrosis and pyroptosis, and its mechanism was mainly associated with the inhibition of reactive oxygen species (ROS)/NLRP3-mediated inflammasome activation.


Introduction
Chronic kidney disease (CKD), a common disease caused by external and internal stimuli, manifests as structural renal damage and dysfunction, which is associated with increased morbidity and usually develops into renal fibrosis [1,2].Accumulation of the extracellular matrix and tubular atrophy have been identified as the main causes of renal fibrosis [3,4].Inflammation, epithelial-mesenchymal transition, and myofibroblast activation contribute to the pathogenesis of renal fibrosis [5,6].However, the underlying mechanisms of renal fibrosis need to be further investigated, and potential solutions for the treatment of renal fibrosis are urgently needed [7,8].The 5/6 nephrectomized rat model is a commonly used model of chronic renal failure that studies mechanisms and drug interventions for renal fibrosis and inflammation [9].Previous studies have demonstrated that the intrarenal renin-angiotensin system (RAS) is activated in 5/6 nephrectomized rats [10].The 5/6 nephrectomized rat model is also associated with increased levels of angiotensin II (Ang II) [11], which is a potent oxidative stress factor that plays a significant role in the development of renal fibrosis and inflammation [12,13].
Traditional Chinese Medicine (TCM) has a rich history of clinical application in the treatment of various chronic diseases in China.One notable TCM is Bushen Huoxue (BSHX) formula, which comprises the following five medicinal plants: Astragalus mongholicus (Huang Qi), Trigonella foenum-graecum L. (Hu Luba), Rheum palmatum L. (Da Huang), Vaccaria segetalis (Wang Buliuxing), and Curcuma phaeocaulis Val.(E Zhu).BSHX is widely used in clinical practice in China to treat chronic renal failure, as it functions to tonify the kidneys and promote blood circulation.In our previous study, BSHX was found to improve renal function in rats subjected to 5/6 nephrectomy [14,15].Based on these previous findings, the present study aimed to further investigate the molecular mechanisms underlying the beneficial effects of BSHX on renal fibrosis.
Pyroptosis is a newly discovered form of programmed cell death characterized by membrane rupture, cell swelling, and caspase-1 activation [16].Pyroptosis is regulated by the gasdermin (GSDM) family of proteins, leading to the secretion of pro-inflammatory cytokines [17].Interleukin-1beta (IL-1β) and IL-18 are two important pro-inflammatory cytokines involved in pyroptosis.As an important upstream protein of the gasdermin D-N-terminal domain (GSDMD-N), inflammasome activation is linked to the maturation of IL-1β and IL-18 via the activation of caspase-1 [18].Furthermore, pyroptosis is positively associated with fibrosis, and inhibition of inflammasome-related proteins can delay fibrosis development [19].Caspase-1-mediated pyroptosis drives renal inflammation and fibrosis [20].NOD-like receptor protein 3 (NLRP3)-caspase-1-GSDMD-N, the classical signaling pathway involved in pyroptosis, is mediated by several factors, including lysosomal permeabilization and reactive oxygen species (ROS) production [21,22].In this study, we aimed to investigate the efficacy and mechanism of action of a medicine (BSHX) with antioxidative and anti-inflammatory functions, but few side effects in alleviating renal fibrosis.

HPLC analysis
The components of BSHX were measured using an Agilent 1100 HPLC system equipped with a quaternary pump, auto-sampler, degasser, automatic thermostatic column compartment, DAD, and LC/MSD Trap XCT ESI mass spectrometer (Agilent Technologies, MA, USA), as previously described [23].

Animal model
Thirty male Sprague-Dawley (SD) rats weighing 150-200 g were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China).Twenty-four rats underwent 5/6 nephrectomy (5/6 Nx), and six rats were assigned to the sham group.After anesthesia with isoflurane/oxygen (1:5), two-thirds of the left kidney was excised.One week later, the right kidney was excised.All rats subjected to 5/6 Nx were randomly assigned to four groups: 5/6 Nx group; 5/6 Nx + 0.25 g/kg BSHX group; 5/6 Nx + 0.5 g/kg BSHX group; 5/6 Nx + 1 g/kg BSHX group.BSHX was administered once daily by oral gavage.After 1 month of treatment, systolic blood pressure was measured using the tail-cuff method (MK-2000; Muromachi Kikai, Tokyo, Japan).All rats were sacrificed after 1 month of treatment.Creatinine, blood urea nitrogen (BUN), and urine protein levels were analyzed using the Creatinine Assay Kit (C011-1-1), Urea Assay Kit (C013-2-1), and Urine Protein Assay Kit (C035-2-1; all from Nanjing Jiancheng Bioengineering Institute, Nanjing, China), respectively, according to the manufacturer's instructions.Renal tissues were immediately excised and fixed with 4% paraformaldehyde or snap-frozen at −80 °C.The protocols were reviewed and approved by the Animal Care Committee of the Seventh People's Hospital affiliated with Shanghai University of Traditional Chinese Medicine (PZSHUTCM220711030).

Histopathology assay
After fixing in 10% formalin for 24 h, renal tissues were embedded in paraffin, cut into slices, deparaffinized with xylene baths, and rehydrated with graded alcohols.Hematoxylin and eosin (H&E) staining was performed as follows: (1) hematoxylin staining for 5 min at 37 °C and (2) 0.5% eosin for 2 min.Masson's trichrome staining was performed according to the manufacturer's protocol (Leagene, Beijing, China).Myofibers are shown in red, while collagen fibers are indicated by light green or aniline blue staining.Images were captured using an optical microscope (Leica).

Cell culture
Human renal proximal tubular epithelial HK-2 cells (Cell Bank of Shanghai Biology Institute, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco) in an incubator (37 °C, 95%, CO 2 :5%).The cells in the vehicle group were cultured in DMEM + vehicle.Cells in the Ang II group were cultured in DMEM with +1 µM Ang II.Cells in the N-acetylcysteine (NAC) group were cultured in DMEM + 1 µM Ang II + 100 µM NAC (Selleck, Radnor, PA, USA), which was used as a positive control.The cells in the BSHX group were cultured in DMEM +1 µM Ang II + BSHX (0.1, 0.2, or 0.5 mg/mL).Finally, cells were collected for further assays after 24 h of treatment.

Cell counting kit-8 assay
Cell viability was determined using a CCK-8 kit (CK04; Dojindo Molecular Technologies, Kumamoto, Japan).Briefly, cells were cultured in a 96-well plate (3 × 10 3 cells per well).Then, 10 μL CCK-8 solution was added to each well, and the reaction time was maintained for 1 h.The absorbance value was obtained by reading the plate using a microplate reader (Pulangxin, Beijing, China) at 450 nm.

Overexpression of NLRP3
A lentivirus targeting NLRP3 was used to overexpress NLRP3 and was purchased from Genechem (Shanghai, China).

Measurement of intracellular ROS production
After treatment with Ang II and BSHX, HK-2 cells were incubated in phosphate buffer saline containing 20 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37 °C for 20 min.Subsequently, the cells were washed with phosphate buffer saline and harvested into tubes.Intracellular ROS production was measured by flow cytometry (BD Biosciences, San Jose, CA, USA).

Cell pyroptosis
Propidium iodide (PI) and caspase-1 p20 staining were used to determine pyroptotic cell death.Cells (5 × 105 cells/well) were cultured in a six-well plate, reached 50% confluence and were then conditioned with caspase-1 p20 antibody/ FITC (Eterlife Ltd., UK) and 10 μl PI (Thermo Fisher) for 15 min.Flow cytometry was used to analyze the samples.

Network pharmacology
The components of BSHX were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database.Targets were searched in GeneCards using the keywords "renal fibrosis, chronic kidney disease".A Venn diagram was drawn to identify the overlapping genes of BSHX and renal fibrosis.The Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform was used for the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses.

Statistical analysis
All data are presented as the mean ± standard deviation (SD).All experiments were performed at least three times.One-way analysis of variance (ANOVA) followed by Dunnett's test for multiple comparisons used, and p < 0.05 indicates statistical significance.

BSHX improves renal function and fibrosis in 5/6 Nx rats
To investigate the functional role of BSHX in regulating renal fibrosis, we used 5/6 Nx SD rats to construct a renal fibrosis model and administered different concentrations of BSHX.H&E and Masson staining showed that 5/6 nephrectomy resulted in severe kidney structural impairment and collagen deposition in rats, which were ameliorated by treatment with BSHX (Figure 2(A)).5/6 Nx significantly increased systolic blood pressure, and BSHX treatment significantly inhibited the increase in systolic blood pressure induced by 5/6 Nx (Figure 2(B)).Moreover, renal function indices, including BUN, creatinine, and urinary protein, were significantly increased in 5/6 Nx rats, which was reversed by BSHX treatment (Figure 2(C-E)).As shown in Figure 2(F-H), 5/6 Nx contributed to increased Hyp levels in renal tissues and serum levels of TGF-β, MMP2, and MMP9, which were improved by treatment with BSHX.To further identify the related signaling pathways that mediate the interaction between BSHX and CKD, we performed a KEGG enrichment analysis of the common targets of BSHX and CKD.The top 10 signaling pathways for KEGG enrichment analysis are shown in Fig S1 .Similarly, 5/6 Nx contributed to the activation of the TGF-β/Smad2/3 signaling pathway in renal tissues, which was improved by treatment with BSHX (Figure 2(I-J)).

BSHX attenuates oxidative stress and NLRP3 inflammasome activation in 5/6 Nx rats
The oxidative stress levels were measured.Rats with 5/6 Nx exhibited higher activity of MDA and lower activities of SOD and GSH than rats with sham surgery, which was corrected by treatment with BSHX (Figure 3(A-C)).As reported previously [19], NLRP3 inflammasome activation is associated with fibrosis, and we aimed to clarify whether an interaction exists between BSHX and NLRP3 inflammasome activation.The results shown in Figure 3(D-F) showed that NLRP3, caspase-1 p20, GSDMD-N, IL-1β, and IL-18 protein levels increased, which decreased after BSHX administration.

BSHX alleviates Ang II-induced fibrosis and oxidative stress in HK-2 cells
To further investigate the role of BSHX in fibrosis and oxidative stress, HK-2 cells were treated with Ang II (1 µM) and different concentrations of BSHX (0.1, 0.2, and 0.5 mg/mL).The CCK-8 assay showed that treatment with 0.5 mg/mL BSHX significantly inhibited the Ang II-induced decrease in cell viability (Figure 4(A)).As shown in Figure 4(B-F), Ang II significantly increased the levels of Hyp, TGF-β, MMP2, and MMP9, along with significant activation of the Smad2/3 signaling pathway, which was inhibited by BSHX treatment.In addition, Ang II increased MDA and ROS levels and decreased antioxidant enzyme activities (GSH and SOD), which were reversed by BSHX treatment (Figure 4(G-K)).
cytometry.As shown in Figure 5(D,E), BSHX treatment alleviated Ang II-induced increase in pyroptosis in HK-2 cells.

BSHX suppresses the NLRP3-mediated fibrosis and pyroptosis in HK-2 cells
We further investigated the role of NLRP3 in the effects of BSHX on fibrosis and pyroptosis in HK-2 cells.Overexpressing lentivirus targeting NLRP3 was applied to HK-2 cells together with 0.5 mg/mL BSHX.As shown in Figure 6(A-E), NLRP3 overexpression increased the levels of Hyp, TGF-β, MMP2, and MMP9 and activated the Smad2/3 signaling pathway; however, these effects were alleviated by BSHX treatment in HK-2 cells.Furthermore, BSHX treatment alleviated the increase in pyroptosis induced by NLRP3 overexpression (Figure 6(F,G)).Consistent with this, NLRP3 overexpression increased the protein levels of NLRP3, caspase-1 p20, GSDMD-N, IL-1β, and IL-18, and these effects were alleviated by BSHX treatment (Figure 6(H-J)).Overall, BSHX regulation of fibrosis and pyroptosis in HK-2 cells is mediated by NLRP3.

Discussion
The 5/6 Nx rat model is a commonly used model of chronic renal failure that studies mechanisms and drug interventions for renal fibrosis and inflammation [9].Our previous studies showed that BSHX protects against renal fibrosis and improves renal function in rats with 5/6 Nx by regulating the expression of TNF-, NF-B, E-cadherin, α-SMA, TGF-β1, CTGF, PPAR, OPN, fibronectin, and laminins [14,15], suggesting an important role of BSHX in alleviating renal fibrosis.Previous studies have demonstrated that the intrarenal RAS is activated in 5/6 Nx rats [10], and the 5/6 Nx rat model is also associated with increased levels of Ang II [11], which is a potent renal growth factor that plays a significant role in the development of fibrosis.The mechanism of Ang II involvement in renal fibrosis is associated with the stimulation of the release of pro-inflammatory and pro-fibrogenic cytokines, which contribute to the accumulation of extracellular matrix and subsequent fibrotic processes [24].Our results suggest that BSHX plays an antioxidant role in combating Ang II-induced oxidative stress and potentially contributes to its protective effects against fibrosis development, further supporting the potential therapeutic value of BSHX in the treatment of renal fibrosis and highlighting its ability to modulate antioxidant mechanisms in renal cells.
Matrix metalloproteinases (MMPs) possess many biological functions, such as tissue remodeling and growth, wound repair, defense mechanisms, and immune responses [25].MMPs can be categorized as collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs.MMP2 and MMP9 are members of the gelatinase subfamily [26].Several studies have shown that ROS accumulation promotes the expression of MMP2, and MMP9 participates in all stages of different forms  of CKD, which is characterized by renal fibrosis [25,27].Consistent with this, increases in both MMP2 and MMP9 were observed in Ang II-treated HK-2 cells, which could be alleviated by treatment with BSHX.Kidney injury is often replaced by renal scarring, which later transforms into renal fibrosis.During the development of renal fibrosis, TGF-β serves as a key regulator of this process by inducing excessive formation of the extracellular matrix.The TGFβ1/SMAD/PAI-1 axis induces renal fibrosis [28].Consistently, Ang II-induced activation of the TGFβ1/Smad pathway was attenuated by BSHX therapy.
In the present study, treatment with BSHX reduced pyroptosis induced by Ang II in a dose-dependent manner, suggesting that BSHX treatment has the potential to alleviate renal fibrosis, possibly by mitigating the occurrence of pyroptosis.Pyroptosis is a recently discovered form of programmed cell death characterized by inflammasome activation and differs from apoptosis and autophagy in terms of cell morphology and function [29].A notable feature of pyroptosis is the release of inflammatory factors, including IL-1β and IL-18.The pathways involved in pyroptosis can be categorized into canonical and non-canonical pathways, which are regulated by caspase-1 and caspase-4/5/11, respectively [30,31].Activation of the caspase-1-mediated canonical pathway leads to upregulated levels of NLRP3 and caspase-1, as well as the release of IL-1β and IL-18 inflammatory bodies [32,33].Notably, the depletion of NLRP3 significantly alleviated the severity of renal fibrosis in mouse models [34,35].Additionally, studies have reported that pyroptosis in renal tubules, mediated by the TNFα/Casp3/GSDME signaling pathway, leads to tubular loss and renal fibrosis [7,33].Our results shed light on the therapeutic value of BSHX in managing renal fibrosis and highlight its potential as a modulator of pyroptotic pathways in kidneys.
combination to observe its efficacy in improving renal fibrosis.The BSHX extract should be analyzed in future studies.Second, a gene-knockout animal would be a better option to verify the effect of BSHX in treating renal fibrosis.Furthermore, transcriptomics would help us to better understand the mechanisms of action of Chinese herbal medicine.These issues will be investigated in future studies.
The evidence presented in this study strongly supports the therapeutic effect of BSHX in inhibiting renal fibrosis in both animal and cellular models.Further studies involving human patients are necessary to investigate the potential clinical application of BSHX.These studies will improve our understanding of the efficacy of BSHX as a clinical treatment for renal fibrosis.

Figure 1 .
Figure 1.HPlC-eSi-MS chromatogram of the BSHX extract in (a) positive and (B) negative mode.